Many biologically active molecules transduce their signals through heptahelical receptors called G protein coupled receptors (GPCRs), which interact specifically with heterotrimeric guanine nucleotide-binding proteins, called G proteins. The interaction of a G protein with its cognate GPCR results in the initiation of intracellular signaling events which, in turn, lead to a variety of important cellular responses (Hamm et al., 1996, Curr. Opin. Cell. Biol. 8: 189-196; Hamm, 1998, Science 273: 669-672). Stimulation of a GPCR by its appropriate agonist causes conformational changes within the receptor that lead to the interaction of the activated receptor with a specific heterotrimeric G protein. Thus, the GPCR-G protein interaction plays an important role in determining the specificity and temporal characteristics of a variety of cellular responses.
Heterotrimeric G proteins are composed of a single xcex1 subunit complexed with the xcex2xcex3 dimer. Molecular cloning has resulted in the identification of 18 distinct xcex1 subunits, 5 xcex2 subunits, and 12 xcex3 subunits. G proteins are usually divided into four subfamilies Gi, Gs, Gq, and G12 based on the sequence similarity of the Gxcex1 subunit. Several lines of evidence suggest that the interaction between a given GPCR and its cognate G protein involves multiple sites of contact on both proteins. All three intracellular loops as well as the carboxyl terminal tail of the receptor have been implicated. The GPCR is thought to interact with all three subunits of the G protein. As the receptor-G protein interaction can be disrupted by a number of treatments that block the carboxyl terminus, including pertussis toxin-catalyzed ADP-ribosylation of Gi and binding of monoclonal antibodies, the carboxy terminal region of the Gxcex1 subunit has been the most intensely investigated contact site. These studies have shown that the Gxcex1 carboxy terminal region is important not only to the interaction, but also plays a critical role in defining receptor specificity (Hamm et al., 1988, Science 241: 832-5; Osawa et al., 1995, J. Biol. Chem. 270: 31052-8; Garcia et al., 1995, EMBO 14: 4460-9; Sullivan et al., 1987, Nature 330: 758-760; Rasenick et al., 1994, J. Biol. Chem. 269: 21519-21525; West et al., 1985, J. Biol. Chem. 260: 14428-30; Conklin et al., 1993, Nature 363: 274-276; Conklin et al., 1996, Mol. Pharmacol. 50: 885-890). Furthermore, it has been shown that peptides corresponding to the carboxy terminal region of a Gxcex1 subunit can block GPCR signaling events (Hamm et al., 1988, Science 241: 832-5; Gilchrist et al., 1998, J. Biol. Chem 273: 14912-19).
Because many medically significant biological processes are mediated by G proteins and their downstream effector molecules, the GPCRs with which they interact, have been the focus of intense drug discovery efforts (Holler et al:, 1999, Cell. Mol. Life Sci. 55: 257-70; Farfel et al., 1999, New Engl. J. Med. 340: 1012-20). A number of therapeutic agents targeting GPCRs have been discovered. Traditionally, the agonist binding site on the GPCR is the point of intervention. However, for some GPCR""s, classical antagonists have been difficult to identify. Such is the case for proteinase activated receptors (PAR), classical antagonists are ineffective due to the unique mechanism of enzymatic cleavage of the receptor and generation of a tethered ligand. In other cases, intrinsic or constitutive activity of receptors directly leads to pathology. Thus, alternative targets for blocking downstream consequences of GPCR signaling are needed. Agents are needed that can inhibit GPCR by blocking the receptor-G protein interface. The present invention satisfies these needs.
The present invention includes an isolated nucleic acid comprising a minigene, wherein said minigene encodes a modified carboxy terminal Gxcex1 peptide, wherein said peptide blocks the site of interaction between a G protein and a G protein coupled receptor in a cell, such as a human cell. In addition, the minigene can further comprise one or more of a promoter, a ribosomal binding site, a translation initiation codon, and a translation termination codon.
In one embodiment, the nucleotide sequence of a minigene of the invention can be one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.
In another embodiment, a minigene encodes a modified carboxy terminal Gxcex1 peptide having one of the following general formulas: MGX, MX, and MZX,
wherein M is a methionine amino acid residue,
wherein G is a glycine amino acid residue,
wherein Z is an amino acid residue other than a glycine amino acid residue, and
wherein X is a carboxy terminal Gxcex1 peptide which comprises an amino acid sequence of the carboxy terminus of a Gxcex1 subunit, and has the property of binding a G protein coupled receptor. In this embodiment, X can comprise from at least about three contiguous amino acids to at least about 54 contiguous amino acids, from at least about three contiguous amino acids to at least about eleven contiguous amino acids, and at least about eleven contiguous amino acids. In one embodiment, X comprises the seven contiguous terminal amino acid residues of the carboxy terminus of a Gxcex1 subunit. For example, the amino acid sequence of a modified carboxy terminal Gxcex1 peptide can be one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29.
The present invention also includes a composition comprising a modified carboxy terminal Gxcex1 peptide having a general formula selected from the group consisting of MGX, MX, and MZX,
wherein M is a methionine amino acid residue,
wherein G is a glycine amino acid residue,
wherein Z is an amino acid residue other than a glycine amino acid residue, and
wherein X is a carboxy terminal Gxcex1 peptide which comprises an amino acid sequence of the carboxy terminus of a Gxcex1 subunit, and has the property of binding a G protein coupled receptor. In this embodiment, X can comprise from at least about three contiguous amino acids to at least about 54 contiguous amino acids, from at least about three contiguous amino acids to at least about eleven contiguous amino acids, and at least about eleven contiguous amino acids. In one embodiment, X comprises the seven contiguous terminal amino acid residues of the carboxy terminus of a Gxcex1 subunit. For example, the amino acid sequence of a modified carboxy terminal Gxcex1 peptide can be one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29.
Additionally, the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one of a modified carboxy terminal Gxcex1 peptide and a minigene encoding a modified carboxy terminal Gxcex1 peptide.
In one aspect, the invention encompasses a method of inhibiting a G protein-mediated signaling event in a cell. This method comprises administering to a cell, preferably a human cell, one of a modified carboxy terminal Gxcex1 peptide, and an isolated nucleic acid comprising a minigene which encodes a modified carboxy terminal Gxcex1 peptide, whereby following the administration, the carboxy terminal Gxcex1 peptide inhibits the G protein mediated signaling event in the cell.
Additionally, the invention includes a method of blocking the site of interaction between a G protein and a G protein coupled receptor in a cell. This method comprises administering to a cell, preferably a human cell, one of a modified carboxy terminal Gxcex1 peptide, and an isolated nucleic acid comprising a minigene which encodes a modified carboxy terminal Gxcex1 peptide, whereby following the administration, the modified carboxy terminal Gxcex1 peptide blocks the site of interaction between the G protein and the G protein coupled receptor in the cell.
Further, the invention includes a method of inhibiting one or more of migration, permeability, and proliferation of a cell. This method comprises administering to a cell, preferably a human cell, one of a modified carboxy terminal Gxcex1 peptide, and an isolated nucleic acid comprising a minigene which encodes a modified carboxy terminal Gxcex1 peptide, wherein the modified carboxy terminal Gxcex1 peptide blocks a G protein-mediated signaling event in a cell, thereby inhibiting one or more of migration, permeability, and proliferation of a cell.
In one embodiment, the preferred cell is a human cell.
In other embodiments, the modified carboxy terminal Gxcex1 peptide, either as administered or as expressed from a minigene of the invention, has a general formula. selected from the group consisting of MGX, MX, and MZX,
wherein M is a methionine amino acid residue,
wherein G is a glycine amino acid residue,
wherein Z is an amino acid residue other than a glycine amino acid residue, and
wherein X is a carboxy terminal Gxcex1 peptide comprising an amino acid sequence of the carboxy terminus of a Gxcex1 subunit, and having the property of binding a G protein coupled receptor. In these embodiments, X can comprise from at least about three contiguous amino acids to at least about 54 contiguous amino acids, from at least about three contiguous amino acids to at least about eleven contiguous amino acids, and at least about eleven contiguous amino acids. In one embodiment, X comprises the seven contiguous terminal amino acid residues of the carboxy terminus of a Gxcex1 subunit. For example, the amino acid sequence of a modified carboxy terminal Gxcex1 peptide can be one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29. As an alternative example, the nucleotide sequence of a minigene can be any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.
The invention encompasses a method of identifying which G protein binds a G protein coupled receptor in a cell. This method comprises administering to the cell an isolated nucleic acid comprising a minigene which encodes a modified carboxy terminal Gxcex1 peptide, and assessing the level of occurrence of a signaling event in the cell. According to this method, the signaling event is associated with the G protein coupled receptor, and a measurable reduction in the level of occurrence of the signaling event in the cell compared with the level of occurrence of the same event in a cell to which the isolated nucleic acid is not administered, is an indication that said modified carboxy terminal Gxcex1 peptide identifies a G protein that binds said G protein coupled receptor.
The invention additionally includes a method of treating a pathological disorder in a mammal. This method comprises administering to the mammal an amount of either a modified carboxy terminal Gxcex1 peptide or an isolated nucleic acid comprising a minigene which encodes a modified carboxy terminal Gxcex1 peptide. According to the method, the amount of either the modified carboxy terminal Gxcex1 peptide or the isolated nucleic acid is sufficient to inhibit a G protein-mediated signaling event associated with the pathological disorder, and thereby alleviate at least one symptom of the pathological disorder.
In one embodiment, the method of treating a pathological disorder in a mammal includes administering to the mammal a modified carboxy terminal Gxcex1 peptide having a general formula selected from the group consisting of MGX, MX, and MZX,
wherein M is a methionine amino acid residue,
wherein G is a glycine amino acid residue,
wherein Z is an amino acid residue other than a glycine amino acid residue, and
wherein X is a carboxy terminal Gxcex1 peptide which comprises an amino acid sequence of the carboxy terminus of a Gxcex1 subunit, and has the property of binding a G protein coupled receptor. In these embodiments, X can comprise from at least about three contiguous amino acids to at least about 54 contiguous amino acids, from at least about three contiguous amino acids to at least about eleven contiguous amino acids, and at least about eleven contiguous amino acids. In one embodiment, X comprises the seven contiguous terminal amino acid residues of the carboxy terminus of a Gxcex1 subunit. For example, the amino acid sequence of a modified carboxy terminal Gxcex1 peptide can be one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29. As an alternative example, the nucleotide sequence of a minigene can be any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.
In multiple embodiments, the method of the invention is useful for treating a pathological disorder selected from the group consisting of stroke, myocardial infarction, restenosis, atherosclerosis, hypotension, hypertension, angina pectoris, acute heart failure, cardiomyocyte apoptosis, cancers, bacterial infections, fungal infections, protozoan infections, viral infections, septic shock, pain, chronic allergic disorders, asthma, inflammatory bowel disease, osteoporosis, rheumatoid arthritis, Grave""s disease, post-operative ileus, diabetes, adult respiratory distress syndrome, myastenia gravis, cardiovascular disease, congestive heart failure, Chagas disease, disorders associated with solid organ transplant, vascular sclerosis, chronic rejection, chronic obstructive pulmonary disease, urinary retention, testotoxicosis, infertility, ulcers, obesity, benign prostatic hypertrophy, anxiety, epilepsy, schizophrenia, manic depression, Parkinson""s disease, Alzheimer""s disease, delirium, dementia, drug addiction, anorexia, and bulimia.
In another aspect, the invention includes a method of treating a disorder associated with an endothelial cell in a mammal, comprising administering to the endothelial cell of the mammal an isolated nucleic acid comprising a minigene, wherein said minigene encodes a modified carboxy terminal Gxcex1 peptide, or a modified carboxy terminal Gxcex1 peptide encoded thereby. According to this method, the peptide blocks a G protein-mediated signaling event in the endothelial cell, and thereby alleviates at least one symptom of the disorder associated with the endothelial cell in the mammal.
The invention additionally includes a composition comprising a peptide having an amino acid sequence selected from the group consisting of SEQ ID Nos: 39, 40, 41, 42, 43, 44, 45, 46, and 47.
Further, the invention includes a method of preventing conception in a female mammal. This method comprises administering to the female mammal either of a modified carboxy terminal Gxcex1 peptide or an isolated nucleic acid comprising a minigene, wherein the minigene encodes a modified carboxy terminal Gxcex1 peptide, thereby preventing conception in the female mammal.